Three monoclonal antibodies (MAbs) were generated from splenocytes of a BALB/c mouse immunized with heat-killed Mycobacterium tuberculosis. All three MAbs bound to surface epitopes of M. tuberculosis as shown by whole-cell enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence, and immunoelectron microscopy. One immunoglobulin M (IgM) MAb bound to lipoarabinomannan, the second IgM MAb bound to mycolyl-arabinogalactan-peptidoglycan complex, and the third MAb, an IgG3, bound to a surface epitope of an uncertain nature. The MAbs demonstrated different cross-reactivity patterns with other mycobacteria. Two of the MAbs were used to develop a modified ELISA spot assay for the detection of mycobacteria.